Lyme disease is a frequently diagnosed human disease and is the most prevalent tick-borne disease in North America, Europe and other parts of the world with a moderate climate. See, A. G. Barbour & D. Fish, Science 260:1610-16 (1993); J. F. Anderson, Rev. Insect Dis. 11:51451-59 (1989); A. C. Steere, N. Engl. J. Med., 331:586-96 (1989). Lyme disease or Lyme borreliosis is a multistage infection caused by Borrelia spirochetes. The Borrelia organism is transmitted to humans and animals by infected Ixodes ticks. White-tailed deer and the white-footed mouse, Peromyscus leucopus, serve as primary reservoirs in nature for the adult tick and larval forms, respectively.
Lyme borreliosis infection in humans and animals causes a number of different clinical manifestations depending upon the stage of the infection. Early infection of humans is usually a flu-like illness with a characteristic skin rash called erythema migrans. The erythema migrans spreads centrifugally and is usually ring-shaped. The erythema migrans usually develops within 1-5 weeks after a tick bite and spontaneously resolves in several weeks or months. See, H. W. Pfister et al., Lancet 343:1013 (1994).
Within a few weeks to several months after infection with Borrelia, the infection may spread to various organs including the brain, nerves, eyes, joints and heart. This spread of infection indicates stage II of the disease is underway. Neurological features of Lyme borreliosis including meningoradiculoneuritis (Bannwarth's syndrome), meningitis, cranial neuritis of the facial nerve, plexus neuritis, mononeuritis multiplex, and, rarely, encephalitis, myelitis, cerebral vasculitis, CSF lymphocyte pleocytosis.
Lyme carditis is a serious condition and commonly features transient atrioventricular block of various degrees, rhythm disturbances, myo-pericarditis and heart failure. Common symptoms of Lyme carditis include palpitations, chest discomfort, shortness of breath, dizziness on exercise and Adams-Stokes attacks.
Lyme borreliosis infection of the musculoskeletal system causes symptoms such as myalgia, arthralgia, arthritis, myositis and lymphadenopathy. Borrelia infection of the eyes produces symptoms such as conjunctivitis, iridocyclitis, choroiditis, optic neuropathy with pupilloedma, panophthalmitis. Infection of other organs may produce hepatomegaly, hepatitis, coughing and testicular swelling.
After months to years of infection, chronic organ involvement may occur indicating the lyme borreliosis has entered Stage III. Symptoms of this stage include chronic arthritis, monarticular arthritis, oligoarticular arthritis, acrodermatitis chronica atrophicans, encephalitis, myositis, keratitis, chronic polyneuropathy and dilated cardiomyopathy.
The Borrelia spirochetes known to cause Lyme borreliosis were originally designated Borrelia burgdorferi, but are now classified into three major genomic species. See, R. T. Marconi & C. F. Guron, J. Clin. Microbiol., 30:2830-34 (1992). One group retains the species designation B. burgdorferi, a second has been designated Borrelia garinii and the third group has not yet been assigned a species name and is referred to as the VS461 group.
Another Borrelia species, B. hermsii, is closely related to B. burgdorferi, but is responsible for a different human disease, relapsing fever. B. hermsii has been characterized as belonging to the same species as B. parheri and B. turicatae by DNA hybridization, although each is specific for a particular arthropod vector (Barberi & Hayes, Microbiol Reviews 50:381-400, 1986). Two other Borrelia species B. anserina and B. coriaceae are closely related to B. burgdorferi, but are not infectious for humans.
Borrelia organisms have a wavy shape and flagella like other spirochetes. A. G. Barbour & S. F. Hayes, Microbiol. Rev., 50:381-400 (1986). These organisms also have a chromosome and several extrachromosomal elements that are linear rather than circular. A. G. Barbour & C. F. Garon, Science, 237:409 (1987). Several surface-exposed lipoproteins, OspA and OspB have been identified and used as antigenic markers in serologic laboratory testing.
Cultivation of Borrelia organisms from body fluids is difficult, making microbiological diagnosis of lyme borreliosis by culturing unsatisfactory. Serological tests to detect B. burgdorferi have been developed including enzyme-linked immunosorbent assay [ELISA], indirect immunofluorescence assay (IFA) and western blotting. See, M. G. Golightly, Am. J. Clin. Pathol., 99:168-74 (1993). However, poor standardization, false-positive and false-negative results do occur with these serologic tests and limit their usefulness. See, Barbour, Ann. Intern. Med., 110:504 (1989). Patients with early (Stage I) or Stage II infections may not yet have developed detectable levels of antibodies and cross reactions with Treponema or other Borrelia not associated with Lyme disease may occur. Treatment with antibiotics may also prevent or delay the development of detectable antibodies in patients with Lyme borreliosis. Together these deficiencies limit the usefulness and reliability of serologic tests in diagnosis and treatment of Lyme borreliosis.
The use of oligonucleotides having specific nucleotide sequences as probes for the recognition of infectious agents is becoming an alternative to the problematic immunological detection assays. At least a portion of both genomic and plasmid DNA sequences of Borrelia have been obtained. See, Schwan et al., J. Clin. Microbiol., 27:1734 (1989); Schwan et al., Ann. N.Y. Acad. Sci., 539:419 (1988). Nucleic acid hybridization probes derived from the linear plasmid of B. burgdorferi were produced and used to identify B. burgdorferi from a number of Borrelia species. However, these probes are inherently limited because their nucleotide sequences are derived from plasmids that may become unstable over time or can be absent from pathogenic Borrelia isolates. Other nucleic acid hybridization probes targeted to specific Borrelia genes are also inherently limited by the degree of evolutionary stability of the targeted gene. See, e.g., Malloy et al., J. Clin. Microbiol., 28:1089 (1990), Lebach et al., J. Clin. Microbiol., 29:731-737 (1991); and Goodman et al., Infect. Immun., 59:269-278 (1991).
Randomly cloned B. burgdorferi DNA sequences were used to construct nucleic acid primers and these primers have been used to amplify target DNA sequences in B. burgdorferi. See, Rosa et al., J. Infect. Dis., 160:1018 (1989). However, not all B. burgdorferi isolates were detected, making these nucleic acid primers unsatisfactory for detection of all B. burgdorferi causing Lyme disease.
The ribosomal RNA (rRNA) genes of B. burgdorferi have been mapped and cloned by Fukunaga and Sohnaka, Biochem. Biophys. Res. Comm., 183:952-57 (1992); and Postic et al., Res. Microbiol., 141:465-475 (1990). B. burgdorferi is unusual in that it appears to contain two copies of the 23S RNA gene and only one copy of the gene encoding 16S rRNA per chromosome. (Fukanaga et al., J. Gen Micobiol. 138:871-877, 1992). The sequence of Borrelia 16S RNA has been used to design hybridization probes that could detect cultured B. burgdorferi organisms. See, Marconi et al., J. Clin. Microbiol., 30:628-32 (1992). The usefulness of the probes to detect B. burdorferi in clinical samples without culturing the organisms was not proven.
To overcome these limitations, nucleic acid amplification of ribosomal RNA sequences has also been described using a broad specificity primer pair to amplify Borrelia 16S rDNA. See, Malloy et al., J. Clin. Microbiol., 28:1089-93 (1990). However, the nucleic acid primers used also amplified S. aureus and P. aeruginosa and thus were not specific for Borrelia burgdorferi.
Four sets of primers derived from 16S rRNA sequences have been used to amplify the DNA of three different Borrelia genomic classes. See, R. T. Marconi and C. F. Baron, J. Clin. Microbiol., 30:2830-34 (1992). Only one primer set, derived from positions 819-842 and 1153-1173 of the Borrelia 16S rRNA, detected all Borrelia organisms present in the various cultured Lyme disease isolates tested. Other primer sets either failed to recognize all three groups of Lyme disease Borrelia or also recognized Borrelia not associated with Lyme disease. The other primer sets failed to amplify all the various Borrelia organisms cultured from the clinical isolates.
The amplification of a 16S rRNA subsequence and of subtypes of Borrelia burgdorferi which vary in the V4 region of the 16S rRNA gene has been described by Adam et al., Infec. Immun., 59:2579-85 (1991). PCR primers have been used to amplify a specific region of 23S rRNA of B. burgdorferi and to distinguish it from other species of Borrelia. See Schwartz et al., J. Clin. Micro. 30:3082 (1992).
Other probes complementary to Borrelia 16S rRNA sequences have been described by Weisburg, EPO Publication No. EPO 0421 725A, Application No. 90310766.2. White and Dodge, PCT US91/01574, disclose primers and probes derived from the 16S rRNA gene of B. burgdorferi and B. hermsii.
Because of the current limitations in serologically detecting and identifying Lyme disease, a need exists for a sensitive procedure to detect all geographical isolates of Borrelia associated with Lyme disease.